This invention relates generally to detection and analysis of particles in fluid samples and in particular to apparatuses and methods for increasing light collection efficiency in capillary based flow cytometry.
Flow cytometry finds a variety of applications throughout the life sciences including clinical diagnostics and immunology, protein and nucleic acid detection, hematology, and oncology. For example, flow cytometry can be used to identify and count particles, cells or microorganisms with specific characteristics in a fluid sample. In a typical flow cytometer, particles to be analyzed are transported in a flowing fluid to an analyzing region where they are illuminated with a focused output beam of a light source. Scatter light and/or fluorescence emitted by the illuminated sample particles are collected and separated according to emission angle and wavelength using optical systems, and detected by photo detectors. The detected scatter light and/or fluorescence in the form of pulses with amplitudes and temporal profiles may be characteristic of the particles' sizes, shapes, and structures etc. A computer system is commonly used to convert analog light signals into digital data streams for subsequent processing and analysis.
The light collection efficiency (CE) of an optical system in a flow cytometer determines the sensitivity and throughput of the instrument. Numerical aperture is a measure of the collection power of an optical system. The collection efficiency increases with the numerical aperture (NA) of the collecting optics as CE˜NA2. Using more powerful, i.e., high NA optics, may provide a straight-forward increase in collection efficiency. However, there is a significant drawback of using high NA optics in capillary based flow cytometry because the sensitivity to a particle position within an analyzing region inside a capillary also increases with the same quadratic dependence on the numerical aperture of the collecting optics (depth-of-field ˜1/NA2), resulting in high coefficient of variation (CV) values or decreased resolution of the instrument. Therefore, in current capillary based flow cytometry in which a capillary bore size defines the area of sample localization for interrogation process, compromises are often empirically found in the configurations of the capillary and light collection optics considering factors such as the usability and availability of the capillary and the desired resolution, sensitivity and manufacturability of the instrument. For instance, in a conventional capillary based cytometer, collecting optics with a numerical aperture of about 0.4-0.5 is used, which provides a collection efficiency of fluorescence about only 3 percent. The low collection efficiency substantially restricts achievable sensitivity and/or throughput of the instrument. With improved air-spaced optics, collection efficiency may increase but is limited by physics to NA=0.69 and collection efficiency to about 10 percent.
Accordingly, there is a need for improved collection efficiency in flow cytometry without significantly compromising the resolution of the instrument. There is a need for increased sensitivity and/or throughput of flow cytometry without significantly compromising the manufacturability or construction cost of the instrument.